Faecal metabolome responses to an altered dietary protein:carbohydrate ratio in adult dogs.

High-protein diets may aid weight loss and weight maintenance programs in both humans and dogs, although the effect of dietary protein levels on gut metabolism and functionality has not been studied in depth. The current study aimed to investigate the effect of an altered dietary protein:carbohydrate ratio on gut function in adult dogs by means of faecal metabolomic fingerprinting. More specifically, functional metabolic differences in dogs fed a high-protein/low-carbohydrate (HPLC) vs. low-protein/high-carbohydrate (LPHC) diet were studied by equally allocating twelve clinically healthy (6 lean and 6 obese) Beagles into two groups in a cross-over design, with each group receiving two isocaloric diets for four weeks. The faecal metabolome revealed that different protein:carbohydrate ratio can influence host and/or gut microbiome metabolism and function, while no effect was observed on the body condition. Targeted analysis demonstrated that the HPLC diet significantly increased the concentration of indole, spermidine, and pipecolinic acid and decreased the concentration of azelaic acid, D-fructose, mannose, and galactose (p < 0.05). Multivariate modelling (OPLS-DA) of the untargeted faecal metabolome revealed distinctly different metabolomic profiles following the HPLC vs. LPHC diet, with 18 altered pathways. The HPLC diet influenced amino acid and lipid metabolism, potentially promoting weight loss and immune function, whereas the LPHC diet affected carbohydrate fermentation and may promote anti-oxidative function.

Exploration and optimization of extraction, analysis and data normalization strategies for mass spectrometry-based DNA adductome mapping and modeling.

Although interest in characterizing DNA damage by means of DNA adductomics has substantially grown, the field of DNA adductomics is still in its infancy, with room for optimization of methods for sample analysis, data processing and DNA adduct identification. In this context, the first objective of this study was to evaluate the use of hydrophilic interaction (HILIC) vs. reversed phase liquid chromatography (RPLC) coupled to high resolution mass spectrometry (HRMS) and thermal acidic vs. enzymatic hydrolysis of DNA followed by DNA adduct purification and enrichment using solid-phase extraction (SPE) or fraction collection for DNA adductome mapping. The second objective was to assess the use of total ion count (TIC) and median intensity (MedI) normalization compared to QC (quality control), iQC (internal QC) and quality control-based robust locally estimated scatterplot smoothing (LOESS) signal correction (QC-RLSC) normalization for processing of the acquired data. The results demonstrate that HILIC compared to RPLC allowed better modeling of the tentative DNA adductome, particularly in combination with thermal acidic hydrolysis and SPE (more valid models, with an average Q2(Y) and R2(Y) of 0.930 and 0.998, respectively). Regarding the need for data normalization and the management of (limited) system instability and signal drift, QC normalization outperformed TIC, MedI, iQC and LOESS normalization. As such, QC normalization can be put forward as the default data normalization strategy. In case of momentous signal drift and/or batch effects however, comparison to other normalization strategies (like e.g. LOESS) is recommended. In future work, further optimization of DNA adductomics may be achieved by merging of HILIC and RPLC datasets and/or application of 2D-LC, as well as the inclusion of Schiff base stabilization and/or fraction collection in the thermal acidic hydrolysis-SPE sample preparation workflow.

Multivariate versus machine learning-based classification of rapid evaporative Ionisation mass spectrometry spectra towards industry based large-scale fish speciation.

Detection and prevention of fish food fraud are of ever-increasing importance, prompting the need for rapid, high-throughput fish speciation techniques. Rapid Evaporative Ionisation Mass Spectrometry (REIMS) has quickly established itself as a powerful technique for the instant in situ analysis of foodstuffs. In the current study, a total of 1736 samples (2015-2021) - comprising 17 different commercially valuable fish species - were analysed using iKnife-REIMS, followed by classification with various multivariate and machine learning strategies. The results demonstrated that multivariate models, i.e. PCA-LDA and (O)PLS-DA, delivered accuracies from 92.5 to 100.0%, while RF and SVM-based classification generated accuracies from 88.7 to 96.3%. Real-time recognition on a separate test set of 432 samples (2022) generated correct speciation between 89.6 and 99.5% for the multivariate models, while the ML models underperformed (22.3-95.1%), in particular for the white fish species. As such, we propose a real-time validated modelling strategy using directly amenable PCA-LDA for rapid industry-proof large-scale fish speciation.

Differences in Metabolic Profiles of Healthy Dogs Fed a High-Fat vs. a High-Starch Diet.

Obesity is a common problem in dogs and overconsumption of energy-rich foods is a key factor. This study compared the inflammatory response and fecal metabolome of dogs fed a high-fat vs. a high-starch diet. Ten healthy lean adult beagles were equally allocated into two groups in a cross-over design. Each group received two diets in which fat (horse fat) and starch (pregelatinized corn starch) were exchanged in an isocaloric way to compare high fat vs. high starch. There was a tendency to increase the glucose and glycine concentrations and the glucose/insulin ratio in the blood in dogs fed with the high-fat diet, whereas there was a decrease in the level of Non-esterified fatty acids and a tendency to decrease the alanine level in dogs fed with the high-starch diet. Untargeted analysis of the fecal metabolome revealed 10 annotated metabolites of interest, including L-methionine, which showed a higher abundance in dogs fed the high-starch diet. Five other metabolites were upregulated in dogs fed the high-fat diet, but could not be annotated. The obtained results indicate that a high-starch diet, compared to a high-fat diet, may promote lipid metabolism, anti-oxidative effects, protein biosynthesis and catabolism, mucosal barrier function, and immunomodulation in healthy lean dogs.

FLEXiGUT: Rationale for exposomics associations with chronic low-grade gut inflammation.

FLEXiGUT is the first large-scale exposomics study focused on chronic low-grade inflammation. It aims to characterize human life course environmental exposure to assess and validate its impact on gut inflammation and related biological processes and diseases. The cumulative influences of environmental and food contaminants throughout the lifespan on certain biological responses related to chronic gut inflammation will be investigated in two Flemish prospective cohorts, namely the "ENVIRONAGE birth cohort", which provides follow-up from gestation to early childhood, and the "Flemish Gut Flora Project longitudinal cohort", a cohort of adults. The exposome will be characterised through biomonitoring of legacy and emerging contaminants, mycotoxins and markers of air pollution, by analysing the available metadata on nutrition, location and activity, and by applying state-of-the-art -omics techniques, including metagenomics, metabolomics and DNA adductomics, as well as the assessment of telomere length and measurement of inflammatory markers, to encompass both exposure and effect. Associations between exposures and health outcomes will be uncovered using an integrated -omics data analysis framework comprising data exploration, pre-processing, dimensionality reduction and data mining, combined with machine learning-based pathway analysis approaches. This is expected to lead to a more profound insight in mechanisms underlying disease progression (e.g. metabolic disorders, food allergies, gastrointestinal cancers) and/or accelerated biological ageing.

Disparities in the gut metabolome of post-operative Hirschsprung's disease patients.

Hirschsprung's disease (HD) is a congenital structural abnormality of the colon seen in approximately 1 to 5000 live births. Despite surgical correction shortly after presentation, up to 60% of patients will express long-term gastrointestinal complaints, including potentially life-threatening Hirschsprung-associated enterocolitis (HAEC). In this study fecal samples from postoperative HD patients (n = 38) and their healthy siblings (n = 21) were analysed using high-resolution liquid chromatography-mass spectrometry aiming to further unravel the nature of the chronic gastrointestinal disturbances. Furthermore, within the patient group, we compared the faecal metabolome between patients with and without a history of HAEC as well as those diagnosed with short or long aganglionic segment. Targeted analysis identified several individual metabolites characteristic for all HD patients as well as those with a history of HAEC and long segment HD. Moreover, multivariate models based on untargeted data established statistically significant (p < 0.05) differences in comprehensive faecal metabolome in the patients' cohort as a whole and in patients with a history of HAEC. Pathway analysis revealed the most impact on amino sugar, lysine, sialic acid, hyaluronan and heparan sulphate metabolism in HD, as well as impaired tyrosine metabolism in HAEC group. Those changes imply disruption of intestinal mucosal barrier due to glycosaminoglycan breakdown and dysbiosis as major metabolic changes in patients' group and should be further explored for potential diagnostic or treatment targets.

Peptidomics of an industrial gluten-free barley malt beer and its non-gluten-free counterpart: Characterisation and immunogenicity.

Recent research suggests that gluten-free beers by prolyl-endopeptidase treatment may not be safe for coeliac disease (CD) patients. Therefore, the gluten peptidome of an industrial gluten-free prolyl-endopeptidase treated malt beer (<10 ppm gluten) was compared to its untreated counterpart (58 ppm gluten) as a reference. NanoLC-HRMS analysis revealed the presence of 155 and 158 gluten peptides in the treated and reference beer, respectively. Characterisation of the peptides in treated beer showed that prolyl-endopeptidase activity was not complete with many peptides containing (multiple) internal proline-residues. Yet, prolyl-endopeptidase treatment did eliminate complete CD-immunogenic motifs, however, 18 peptides still contained partial, and potentially unsafe, motifs. In the reference beer respectively 7 and 37 gluten peptides carried (multiple) complete and/or partial CD-immunogenic motifs. Worrying is that many of these partial immunogenic gluten peptides do not contain a recognition epitope for the R5-antibody and would be overlooked in the current ELISA analysis for gluten quantification.

Valorisation of tainted boar meat in patties, frankfurter sausages and cooked ham by means of targeted dilution, cooking and smoking.

Because of the need to abolish the castration of piglets without anaesthesia/analgesia, the pig industry is searching for a mode of action for the valorisation of meat with boar taint, an off-odour in entire male pigs. Carcasses with boar taint were selected by means of sensory and chemical analysis, after which patties with different levels of tainted boar meat were produced, as well as cooked ham and Frankfurter sausages using different smoke condensates and cooking temperatures. For these products orthonasal and retronasal boar taint odour were assessed by a trained expert panel. The results offer guidance regarding dilution of tainted meat (with <400 µg/kg androstenone if skatole is low or <200 µg/kg androstenone in concurrence with ≥37 µg/kg skatole) and the potential application of smoke condensates (e.g., Rudinsmoke C for sausages and Smokez LFBN for ham) as promising boar taint masking strategies.

Untargeted Metabolomics to Reveal Red versus White Meat-Associated Gut Metabolites in a Prudent and Western Dietary Context.

SCOPE: To improve understanding of the epidemiological link between red and processed meat consumption and chronic diseases, more insight into the formation of metabolites during meat digestion is warranted. METHODS AND RESULTS: Untargeted mass-spectrometry-based metabolomics is applied to explore the impact of red and processed meat consumption (compared to chicken), combined with a prudent or Western dietary pattern. A pig feeding study (n = 32), as a sentinel for humans, is conducted in a 2 × 2 factorial design for 4 weeks. The luminal content of the small intestine and colon are collected to determine their metabolic fingerprints. Seventy-six metabolites (38 in the small intestine, 32 in the colon, and 6 in both intestinal compartments) contributing to the distinct gut metabolic profiles of pigs fed either chicken or red and processed meat are (tentatively) identified. Consumption of red and processed meat results in higher levels of short- and medium-chain acylcarnitines and 3-dehydroxycarnitine, irrespective of dietary context, whereas long-chain acylcarnitines and monoacylglycerols are associated with the red and processed Western diet. CONCLUSION: The identification of red and processed meat-associated gut metabolites in this study contributes to the understanding of meat digestion in a complex but controlled dietary context and its potential health effects.

Nutrimetabolomics: An Integrative Action for Metabolomic Analyses in Human Nutritional Studies.

The life sciences are currently being transformed by an unprecedented wave of developments in molecular analysis, which include important advances in instrumental analysis as well as biocomputing. In light of the central role played by metabolism in nutrition, metabolomics is rapidly being established as a key analytical tool in human nutritional studies. Consequently, an increasing number of nutritionists integrate metabolomics into their study designs. Within this dynamic landscape, the potential of nutritional metabolomics (nutrimetabolomics) to be translated into a science, which can impact on health policies, still needs to be realized. A key element to reach this goal is the ability of the research community to join, to collectively make the best use of the potential offered by nutritional metabolomics. This article, therefore, provides a methodological description of nutritional metabolomics that reflects on the state-of-the-art techniques used in the laboratories of the Food Biomarker Alliance (funded by the European Joint Programming Initiative "A Healthy Diet for a Healthy Life" (JPI HDHL)) as well as points of reflections to harmonize this field. It is not intended to be exhaustive but rather to present a pragmatic guidance on metabolomic methodologies, providing readers with useful "tips and tricks" along the analytical workflow.

Biomarkers of meat and seafood intake: an extensive literature review.

Meat, including fish and shellfish, represents a valuable constituent of most balanced diets. Consumption of different types of meat and fish has been associated with both beneficial and adverse health effects. While white meats and fish are generally associated with positive health outcomes, red and especially processed meats have been associated with colorectal cancer and other diseases. The contribution of these foods to the development or prevention of chronic diseases is still not fully elucidated. One of the main problems is the difficulty in properly evaluating meat intake, as the existing self-reporting tools for dietary assessment may be imprecise and therefore affected by systematic and random errors. Dietary biomarkers measured in biological fluids have been proposed as possible objective measurements of the actual intake of specific foods and as a support for classical assessment methods. Good biomarkers for meat intake should reflect total dietary intake of meat, independent of source or processing and should be able to differentiate meat consumption from that of other protein-rich foods; alternatively, meat intake biomarkers should be specific to each of the different meat sources (e.g., red vs. white; fish, bird, or mammal) and/or cooking methods. In this paper, we present a systematic investigation of the scientific literature while providing a comprehensive overview of the possible biomarker(s) for the intake of different types of meat, including fish and shellfish, and processed and heated meats according to published guidelines for biomarker reviews (BFIrev). The most promising biomarkers are further validated for their usefulness for dietary assessment by published validation criteria.

A validated multi-matrix platform for metabolomic fingerprinting of human urine, feces and plasma using ultra-high performance liquid-chromatography coupled to hybrid orbitrap high-resolution mass spectrometry.

In recent years, metabolomics has surfaced as an innovative research strategy in human metabolism, whereby selection of the biological matrix and its inherent metabolome is of crucial importance. However, focusing on a single matrix may imply that relevant molecules of complementary physiological pathways, covered by other matrices, are missed. To address this problem, this study presents a unique multi-matrix platform for polar metabolic fingerprinting of feces, plasma and urine, applying ultra-high performance liquid-chromatography coupled to hybrid quadrupole-Orbitrap high-resolution mass spectrometry, that is able to achieve a significantly higher coverage of the system's metabolome and reveal more significant results and interesting correlations in comparison with single-matrix analyses. All three fingerprinting approaches were proven 'fit-for-purpose' through extensive validation in which a number of endogenous metabolites were measured in representative quality control samples. For targeted and untargeted validation of all three matrices, excellent linearity (coefficients of determination R2 ≥ 0.99 or 0.90 respectively), recovery and precision (coefficients of variance ≤ 15% or 30% respectively) were observed. The potential of the platform was demonstrated by subjecting fecal, urine and plasma samples (collected within one day) from ten healthy volunteers to metabolic fingerprinting, yielding respectively 9 672, 9 647, and 6122 components. Orthogonal partial least-squares discriminant analysis provided similar results for feces and plasma to discriminate according to gender (p-value, R2(X), R2(Y) and Q2(Y)), suggesting feces as an excellent alternative biofluid to plasma. Moreover, combining the different matrices improved the model's predictivity, indicating the superiority of multi-matrix platforms for research purposes in biomarker detection or pathway elucidation and in the selection of the most optimal matrix for future clinical purposes.

DNA adduct profiling of in vitro colonic meat digests to map red vs. white meat genotoxicity.

The consumption of red meat has been linked to an increased colorectal cancer (CRC) risk. One of the major hypotheses states that heme iron (present in red meat) stimulates the formation of genotoxic N-nitroso compounds (NOCs) and lipid peroxidation products (LPOs). By means of DNA adductomics, chemically induced DNA adduct formation can be mapped in relation to e.g. dietary exposures. In this study, this state-of-the-art methodology was used to investigate alkylation and (lipid per)oxidation induced DNA adduct formation in in vitro red vs. white meat digests. In doing so, 90 alkylation and (lipid per)oxidation induced DNA adduct types could be (tentatively) identified. Overall, 12 NOC- and/or LPO-related DNA adduct types, i.e. dimethyl-T (or ethyl-T), hydroxymethyl-T, tetramethyl-T, methylguanine (MeG), guanidinohydantoin, hydroxybutyl-C, hydroxymethylhydantoin, malondialdehyde-x3-C, O6-carboxymethylguanine, hydroxyethyl-T, carboxyethyl-T and 3,N4-etheno-C were singled out as potential heme-rich meat digestion markers. The retrieval of these DNA adduct markers is in support of the heme, NOC and LPO hypotheses, suggesting that DNA adduct formation may indeed contribute to red meat related CRC risk.

DNA adductomics to study the genotoxic effects of red meat consumption with and without added animal fat in rats.

Digestion of red and processed meat has been linked to the formation of genotoxic N-nitroso compounds (NOCs) and lipid peroxidation products (LPOs) in the gut. In this study, rats were fed a meat based diet to compare the possible genotoxic effects of red vs. white meat, and the interfering role of dietary fat. To this purpose, liver, duodenum and colon DNA adductomes were analyzed with UHPLC-HRMS. The results demonstrate that the consumed meat type alters the DNA adductome; the levels of 22 different DNA adduct types significantly increased upon the consumption of beef (compared to chicken) and/or lard supplemented beef or chicken. Furthermore, the chemical constitution of the retrieved DNA adducts hint at a direct link with an increase in NOCs and LPOs upon red (and processed) meat digestion, supporting the current hypotheses on the causal link between red and processed meat consumption and the development of colorectal cancer.

Untargeted metabolomics of colonic digests reveals kynurenine pathway metabolites, dityrosine and 3-dehydroxycarnitine as red versus white meat discriminating metabolites.

Epidemiological research has demonstrated that the consumption of red meat is an important risk factor for the development of colorectal cancer (CRC), diabetes mellitus and cardiovascular diseases. However, there is no holistic insight in the (by-) products of meat digestion that may contribute to disease development. To address this hiatus, an untargeted mass spectrometry (MS)-based metabolomics approach was used to create red versus white meat associated metabolic fingerprints following in vitro colonic digestion using the fecal inocula of ten healthy volunteers. Twenty-two metabolites were unequivocally associated with simulated colonic digestion of red meat. Several of these metabolites could mechanistically be linked to red meat-associated pathways including N'-formylkynurenine, kynurenine and kynurenic acid (all involved in tryptophan metabolism), the oxidative stress marker dityrosine, and 3-dehydroxycarnitine. In conclusion, the used MS-based metabolomics platform proved to be a powerful platform for detection of specific metabolites that improve the understanding of the causal relationship between red meat consumption and associated diseases.

Mass Spectrometric Mapping of the DNA Adductome as a Means to Study Genotoxin Exposure, Metabolism, and Effect.

Covalent binding of endo- or exogenous chemicals to DNA results in the formation of DNA adducts which are reflective of exposure of the human body to DNA-damaging molecules and their metabolic pathways. The study of DNA adduct types and levels in human tissue therefore offers an interesting tool in several fields of research, including toxicology and cancer epidemiology. Over the years, a range of techniques and methods have been developed to study the formation of endo- and exogenous DNA adducts. However, for the simultaneous detection, identification and quantification of both known and unknown DNA adducts, mass spectrometry (MS) is deemed to be the most promising technique. In this perspective, we focus on the analysis of multiple DNA adducts within a sample with the emphasis on untargeted analysis. The advantageous use of MS methodologies for DNA adductome mapping is discussed comprehensively with relevant field examples. In addition, several aspects of study design, sample pretreatment, and analysis are addressed as these factors significantly affect the reliability of DNA adductomics studies.

Diet-related DNA adduct formation in relation to carcinogenesis.

The human diet contributes significantly to the initiation and promotion of carcinogenesis. It has become clear that the human diet contains several groups of natural foodborne chemicals that are at least in part responsible for the genotoxic, mutagenic, and carcinogenic potential of certain foodstuffs. Electrophilic chemicals are prone to attack nucleophilic sites in DNA, resulting in the formation of altered nucleobases, also known as DNA adducts. Since DNA adduct formation is believed to signal the onset of chemically induced carcinogenesis, the DNA adduct-inducing potential of certain foodstuffs has been investigated to gain more insight into diet-related pathways of carcinogenesis. Many studies have investigated diet-related DNA adduct formation. This review summarizes work on known or suspected dietary carcinogens and the role of DNA adduct formation in hypothesized carcinogenesis pathways.

In vitro DNA adduct profiling to mechanistically link red meat consumption to colon cancer promotion.

Colorectal cancer (CRC) is the third most common cancer type in the world. Epidemiological research has demonstrated that both red and processed meat consumption significantly contribute to CRC risk. In this study, red meat toxicity was investigated by means of simulated gastrointestinal conditions, malondialdehyde (MDA) analysis and UHPLC-(HR)MS(/MS) based DNA adductomics. Since dairy products with high calcium content are associated with a decreased CRC-risk, the possible CRC-protective effects of calcium were assessed as well. The obtained results confirmed the earlier reported finding that heme-rich meat stimulates lipid peroxidation and O6-carboxymethylguanine (O6-CMG) DNA adduct formation during digestion. Calcium carbonate (CaCO3) supplementation resulted in both toxic and anti-toxic effects; i.e. stimulation of O6-CMG production, but reduction of MDA formation. DNA adductome mapping of meat digests revealed a significant interindividual variability. The observed DNA adduct profile also differed according to the digested meat type, uncovering different putative DNA adducts that seem to be associated with digestion of beef or chicken with or without supplemented CaCO3. Formamidopyrimidine-adenine was found to be discriminative for meat digests without added CaCO3, carboxyethylcytosine was significantly higher in beef digests and methoxymethylcytosine (or its hydroxyethylcytosine isomer) was found to be lower in meat digests supplemented with CaCO3. These results demonstrate that DNA adduct formation may be involved in the pathway that links red meat digestion to CRC promotion. In addition, the possible CRC-protective attributes of calcium through anti-oxidant actions could be documented.

High resolution mass spectrometry based profiling of diet-related deoxyribonucleic acid adducts.

Exposure of DNA to endo- and exogenous DNA binding chemicals can result in the formation of DNA adducts and is believed to be the first step in chemically induced carcinogenesis. DNA adductomics is a relatively new field of research which studies the formation of known and unknown DNA adducts in DNA due to exposure to genotoxic chemicals. In this study, a new UHPLC-HRMS(/MS)-based DNA adduct detection method was developed and validated. Four targeted DNA adducts, which all have been linked to dietary genotoxicity, were included in the described method; O(6)-methylguanine (O(6)-MeG), O(6)-carboxymethylguanine (O(6)-CMG), pyrimidopurinone (M1G) and methylhydroxypropanoguanine (CroG). As a supplementary tool for DNA adductomics, a DNA adduct database, which currently contains 123 different diet-related DNA adducts, was constructed. By means of the newly developed method and database, all 4 targeted DNA adducts and 32 untargeted DNA adducts could be detected in different DNA samples. The obtained results clearly demonstrate the merit of the described method for both targeted and untargeted DNA adduct detection in vitro and in vivo, whilst the diet-related DNA adduct database can distinctly facilitate data interpretation.

Increased oxidative and nitrosative reactions during digestion could contribute to the association between well-done red meat consumption and colorectal cancer.

Uncured and nitrite-cured pork were subjected, raw, cooked (65 °C, 15 min) or overcooked (90 °C, 30 min), to an in vitro digestion model, which includes mouth, stomach, duodenum, and colon phases. Heating of uncured meat resulted in a pronounced increase in lipid and protein oxidation products throughout digestion. Nitrite-curing had an antioxidant effect during digestion, but this effect disappeared when the meat was overcooked, resulting in up to ninefold higher 4-hydroxy-2-nonenal concentrations compared with digested nitrite-cured raw and cooked pork. Colonic digesta contained significantly higher concentrations of the NOC-specific DNA adduct O(6)-carboxy-methylguanine when pork underwent a more intense heating procedure, independent of nitrite-curing, depending strongly on the fecal inoculum used. Since processed meats are usually nitrite-cured, the present study suggests that overcooking processed meat is likely to result in the formation of genotoxic compounds during digestion and should, therefore, be avoided.